Organization and structure of actin filament bundles in Listeria-infected cells

During its motion inside host cells, Listeria monocytogenes promotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria-column interface. To get a measure of filament organization in the column, Listeria grown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in the Listeria-associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: 1) the surface of the bacteria, 2) the cytoplasm next to the bacteria, 3) the outer shell of the actin column, and 4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant of Listeria that can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility of Listeria monocytogenes inside infected cells.